Cells activate specific signaling pathways in order to transiently adapt to and eventually correct hypoxic stress (Gordan and Simon, 2007; Liu and Simon, 2004). Solid tumors depend on neovascularization for maintained growth (Hanahan and Folkman, 1996) and tumor cells secrete a number of growth and angiogenic factors that stimulate endothelial cell proliferation and, ultimately, the formation of new tumor-associated blood vessels. The Hypoxia Inducible transcription Factor (HIF) is a central regulator of the cellular response to hypoxia (Semenza, 2000) and secreted growth and angiogenic factors, including vascular endothelial growth factor (VEGF), platelet derived growth factor (PDFG), transforming growth factor (TGFa) and angiopoetins are bona fide HIF target genes (Maxwell et al., 2001).
HIF is a heterodimeric transcription factor consisting of hypoxia-regulated (HIF-a) and a constitutively expressed (HIF-1b) subunits (Semenza, 2000). There are two transactivating HIF-a isoforms, HIF-1a and HIF-2a, whose cellular activity is tightly regulated by oxygen (Gordan and Simon, 2007; Raval et al., 2005). In well-oxygenated cells, the tumor suppressor protein pVHL targets HIF-a for ubiquitination and proteasomal degradation (Maxwell et al., 1999; Ohh et al., 2000). This interaction requires hydroxylation of HIF-a proline by cellular HIF prolylhydroxylases, termed EGLN1, 2 and 3 (Epstein et al., 2001; Ivan et al., 2001; Jaakkola et al., 2001). EGLNs are non-heme, Fe(II) and 2-oxoglurarate dependent dioxygenases (Epstein et al., 2001; Schofield and Ratcliffe, 2004). A decline in intracellular oxygen prevents HIF-a prolyl hydroxylation and disrupts HIF-pVHL interaction. Stabilized HIF-a subunits enter the nucleus, heterodimerize with HIF-1b and bind to DNA sequences termed Hypoxia Response Elements (HREs) to transactivate a large number of hypoxia inducible genes. HIF's transcriptional activity is similarly attenuated by Factor Inhibiting HIF (FIH), a second Fe(II) and 2-oxoglurarate-dependent dioxygenase that hydroxylates a conserved asparagine residue in the transactivation domain of HIF (Bruick and McKnight, 2001; Lando et al., 2002a; Lando et al., 2002b; Mahon et al., 2001). Hypoxia therefore promotes both the stability and transcriptional activity of HIF.
Mutations in cellular proteins that regulate HIF stability and transactivation have been causally linked to cancer development. Inactivation of the tumor suppressor protein pVHL leads to constitutive HIF overexpression, which is both necessary and sufficient for growth of VHL deficient tumors (Kondo et al., 2003; Kondo et al., 2002; Maranchie et al., 2002; Zimmer et al., 2004). Mutations that activate the phosphatidylinositol 3-kinase (PI3K) signaling pathway result in enhanced mTOR activity (Brugarolas et al., 2004; Hudson et al., 2002; Majumder et al., 2004). This likely explains the observation that loss-of-function mutations in PTEN and TSC1/2 genes or Her2/neu overexpression lead to increased HIF expression in normoxic and hypoxic conditions (Brugarolas et al., 2003; Laughner et al., 2001; Zundel et al., 2000).
Part of the adaptive response to the hypoxia in healthy cells is to conserve energy when oxidative phosphorylation is restricted via a global decrease in protein translation (Arsham et al., 2003; Bert et al., 2006; Lang et al., 2002; Liu et al., 2006; Schepens et al., 2005). This is, at least in part, mediated by Redd1, itself a HIF target gene, inhibiting mTOR via the tuberous sclerosis (TSC1/2) complex (DeYoung et al., 2008). However, specific messages that are required to synthesize proteins that allow cells to cope with the hypoxic environment are spared this translational repression (Blais et al., 1994; Liu and Simon, 2004; Spicher et al., 1998; Thomas and Johannes, 2007; Wouters et al., 2005). The mechanisms for selectively supporting translation of certain messages in conditions of hypoxia are under investigation.
The present invention is directed to overcoming these and other deficiencies in the art.